Recombinant Anti-KIF5B antibody [EPR10277(B)] (ab151558)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: KIF5B knockout HAP1 whole cell lysate (20 µg)
Lane 3: Hek293 whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab151558 observed at 110 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab151558 was shown to specifically react with KIF5B in wild type cells as signal was lost in KIF5B knockout cells. Wild-type and KIF5B knockout samples were subjected to SDS-PAGE. Ab151558 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HeLa cells stained with ab151558 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab151558, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling KIF5B with ab151558 at 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human uterus tissue labeling KIF5B with ab151558 at 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.